Project Summary/Abstract Many viruses use non-coding RNAs to manipulate host cell processes. An example of one such RNA is found in arthropod-borne flaviviruses (FVs), a class of virus responsible for serious public health threats including Dengue, Yellow Fever, and West Nile Virus. FVs produce subgenomic flavivirus RNAs (sfRNAs), RNA fragments about 200-500 nts in length which have been found to be responsible for the pathology of FV infection in model infection systems. sfRNA fragments are generated by cleavage of the full-length flaviviral genome by the endogenous 5??3? exonuclease Xrn1. Specific structured RNA elements within the sfRNA resist Xrn1 degradation, halting Xrn1 cleavage and leaving the sfRNA fragment behind. These structured RNA elements are known as Xrn1-resistant RNAs (xrRNAs), and mutations to xrRNAs are found to eliminate the accumulation of sfRNA fragments during flavivirus infection. A structural study in the Kieft laboratory has identified the three-dimensional fold of an xrRNA element. The folding of the RNA hints at potential mechanisms for Xrn1 resistance, however it is currently unclear whether the RNA forms a static, highly-stable RNA structure or accomplishes its activity through dynamic interactions with the Xrn1 entrance channel. A full mechanistic understanding of xrRNAs requires an understanding of the stability and molecular dynamics of xrRNA folding. In my first aim, I propose to use single-molecule Frster resonance energy transfer (FRET) combined with quantitative biochemical assays of Xrn1 resistance in order to quantify folding transitions in xrRNAs and demonstrate how xrRNA folding contributes to Xrn1 resistance. There are several distinct lineages of arthropod-bourne FVs, including mosquito-bourne (MB), tick-bourne (TB), no known vector (NKV), and insect specific flaviviruses (ISF). The sfRNA sequences derived from these lineages are highly divergent, therefore further study of TB, NKV, and ISF lineage sfRNA is required to determine how they resist Xrn1 degradation. In my second aim, I propose to biochemically characterize the xrRNA elements from TB, NKV, and ISF lineages in order to determine which regions of the sfRNA are responsible for Xrn1 resistance and determine their secondary structure. I also propose to determine the three- dimensional structure of these elements by X-ray crystallography. sfRNAs have been found to inhibit the activity of Xrn1 by forming a complex with Xrn1 and sequestering it, altering the regulation of RNA transcripts during infection. In my third aim I propose to further study this effect. I have created a quantitative Xrn1 resistance assay in order to determine the minimal RNA sequence requirements for Xrn1 sequestration activity and identify how sfRNAs interact with Xrn1.